Since the advent of structural biology in the 1960's, the general strategy for illuminating the physical basis of the function of a biological macromolecule has been to correlate the observed changes in a particular biological process with accompanying structural changes. Until recently nearly all studies in biochemistry considered a protein native state as a single species, commonly represented by the high-resolution structure obtained by NMR spectroscopy or X-ray crystallography. A variety of experimental and theoretical perspectives have promoted a broadening of this view to include an ensemble of interconverting conformational states. The core goal of this proposal is to understand the structures and energetics of the ensemble of states that a protein explores under native conditions and to illuminate the role of these states in a variety of fundamental processes. For example, a critical prediction of the modern thermodynamic view of protein structure and stability is that proteins should undergo a cold-induced denaturation. Unfortunately, it has been difficult to directly characterize the process of cold denaturation. We show below that cold denaturation of ubiquitin can be followed by high resolution NMR if the protein is encapsulated in a protective reverse micelle and dissolved in a low viscosity fluid. This allows access to temperatures as low as -35 degrees C. The initial results are startling and promote a more detailed examination of the structural and dynamic characteristics of the ensemble of cold induced states. A variety of high resolution NMR methods will be employed to achieve this. In addition, we will also characterize the cold denaturation of apocytochrome b562 and cytochrome c, both model systems for the native state hydrogen exchange technique. These studies should help reveal the network of non-covalent interactions that leads to the remarkable cooperative substructure of proteins and, in turn, should begin to illuminate how proteins use the various states available to them. The ensemble of states will be further probed by use of a complementary native state hydrogen exchange approach that uses hydrostatic pressure as a perturbation, and will also involve examination of the apparent ability of osmolytes to counteract the destabilization by high pressure. Finally, recent theoretical work points to a possible role for fast motion in the promotion of electron transfer by proteins. This potential role will be directly examined in cytochrome c using deuterium and nitrogen-15 relaxation methods.